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Coronaviruses cause respiratory and gastrointestinal diseases in diverse host species. Deltacoronaviruses (DCoVs) have been identified in various songbird species and in leopard cats in China. In 2009, porcine deltacoronavirus (PDCoV) was detected in fecal samples from pigs in Asia, but its etiologic role was not identified until 2014, when it caused major diarrhea outbreaks in swine in the United States. Studies have shown that PDCoV uses a conserved region of the aminopeptidase N protein to infect cell lines derived from multiple species, including humans, pigs, and chickens. Because PDCoV is a potential zoonotic pathogen, investigations of its prevalence in humans and its contribution to human disease continue. We report experimental PDCoV infection and subsequent transmission among poultry. In PDCoV-inoculated chicks and turkey poults, we observed diarrhea, persistent viral RNA titers from cloacal and tracheal samples, PDCoV-specific serum IgY antibody responses, and antigen-positive cells from intestines.

Hepatitis E virus (HEV) is an important emerging pathogen producing significant morbidity in immunosuppressed patients. HEV has been detrimental to solid organ transplant (SOT) patients, cancer patients, and HIV-positive patients, where chronic HEV infections occur. Blood-borne transfusions and multiple cases of chronic HEV infection in transplant patients have been reported in the past few decades, necessitating research on HEV pathogenesis using immunosuppressed animal models. Numerous animal species with unique naturally occurring HEV strains have been found, several of which have the potential to spread to humans and to serve as pathogenesis models. Host immunosuppression leads to viral persistence and chronic HEV infection allows for genetic adaptation to the human host creating new strains with worse disease outcomes. Procedures necessary for SOT often entail blood transfusions placing immunosuppressive patients into a “high risk group” for HEV infection. This scenario requires an appropriate immunosuppressive animal model to understand disease patterns in these patients. Hence, this article reviews the recent advances in the immunosuppressed animal models for chronic HEV infection with emphasis on pathogenesis, immune correlates, and the liver pathology associated with the chronic HEV infections.

One of the most intriguing issues in the hepatitis E virus (HEV) field is the significant increase in mortality rates of the mother and fetus when infection occurs in the second and third trimesters of gestation. A virus that is normally self-limiting and has a mortality rate of less than one percent in otherwise healthy individuals steeply rises by up to 30% in these pregnant populations. Answering this pivotal question has not been a simple task. HEV, in general, has been a difficult pathogen to understand in the laboratory setting. A historical lack of ability to efficiently propagate the virus in tissue culture models has led to many molecular aspects of the viral lifecycle being understudied. Although great strides have been made in recent years to adapt viruses to cell culture, this field remains behind other viruses that are much easier to replicate efficiently in vitro. Some of the greatest discoveries regarding HEV have come from using animal models for which naturally occurring strains of HEV have been identified, including pigs and chickens, but key limitations have made animal models imperfect for studying all aspects of human HEV infections. In addition to the difficulties working with HEV, pregnancy is a very complicated biological process with an elaborate interplay between many different host systems, including hormones, cardiovascular, kidneys, respiratory, gastrointestinal, epithelial, liver, metabolic, immune, and others. Significant differences between the timing and interplay of these systems are notable between species, and making direct comparisons between animals and humans can be difficult at times. No simple answer exists as to how HEV enhances mortality in pregnant populations. One of the best approaches to studying HEV in pregnancy is likely a combinatorial approach that uses the best combination of emerging in vitro and in vivo systems while accounting for the deficiencies that are present in each model. This review describes many of the current HEV animal model systems and the strengths and weaknesses of each as they apply to HEV pregnancy-associated mortality. We consider factors that are critical to analyzing HEV infection within the host and how, despite no perfect animal model for human pregnancy mortality existing, recent developments in HEV models, both in vitro and in vivo, are advancing our overall understanding of HEV in the pregnant host.

Hepatitis E virus is an important emerging pathogen producing a lethal impact on the pregnant population and immunocompromised patients. Starting in 1983, it has been described as the cause for acute hepatitis transmitted via the fecal–oral route. However, zoonotic and blood transfusion transmission of HEV have been reported in the past few decades, leading to the detailed research of HEV pathogenesis. The reason behind HEV being highly virulent to the pregnant population particularly during the third trimester, leading to maternal and fetal death, remains unknown. Various host factors (immunological, nutritional, hormonal) and viral factors have been studied to define the key determinants assisting HEV to be virulent in pregnant and immunocompromised patients. Similarly, chronic hepatitis is seen particularly in solid organ transplant patients, resulting in fatal conditions. This review describes recent advances in the immunopathophysiology of HEV infections in general, pregnant, and immunocompromised populations, and further elucidates the in vitro and in vivo models utilized to understand HEV pathogenesis.

Background Unlike the injectable vaccines, intranasal lipid nanoparticle (NP)-based adjuvanted vaccine is promising to protect against local infection and viral transmission. Infection of ferrets with SARS-CoV-2 results in typical respiratory disease and pathology akin to in humans, suggesting that the ferret model may be ideal for intranasal vaccine studies. Results We developed SARS-CoV-2 subunit vaccine containing both Spike receptor binding domain (S-RBD) and Nucleocapsid (N) proteins (NP-COVID-Proteins) or their mRNA (NP-COVID-mRNA) and NP-monosodium urate adjuvant. Both the candidate vaccines in intranasal vaccinated aged ferrets substantially reduced the replicating virus in the entire respiratory tract. Specifically, the NP-COVID-Proteins vaccine did relatively better in clearing the virus from the nasal passage early post challenge infection. The immune gene expression in NP-COVID-Proteins vaccinates indicated increased levels of mRNA of IFNα, MCP1 and IL-4 in lungs and nasal turbinates, and IFNγ and IL-2 in lungs; while proinflammatory mediators IL-1β and IL-8 mRNA levels in lungs were downregulated. In NP-COVID-Proteins vaccinated ferrets S-RBD and N protein specific IgG antibodies in the serum were substantially increased at both day post challenge (DPC) 7 and DPC 14, while the virus neutralizing antibody titers were relatively better induced by mRNA versus the proteins-based vaccine. In conclusion, intranasal NP-COVID-Proteins vaccine induced balanced Th1 and Th2 immune responses in the respiratory tract, while NP-COVID-mRNA vaccine primarily elicited antibody responses. Conclusions Intranasal NP-COVID-Proteins vaccine may be an ideal candidate to elicit increased breadth of immunity against SARS-CoV-2 variants.

Avian species often serve as transmission vectors and sources of recombination for viral infections due to their ability to travel vast distances and their gregarious behaviors. Recently a novel deltacoronavirus (DCoV) was identified in sparrows. Sparrow deltacoronavirus (SpDCoV), coupled with close contact between sparrows and swine carrying porcine deltacoronavirus (PDCoV) may facilitate recombination of DCoVs resulting in novel CoV variants. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from sparrow coronaviruses (SpCoVs) may enhance infection in poultry. We used recombinant chimeric viruses, which express S protein or the RBD of SpCoV (icPDCoV-SHKU17, and icPDCoV-RBDISU) on the genomic backbone of an infectious clone of PDCoV (icPDCoV). Chimeric viruses were utilized to infect chicken derived DF-1 cells, turkey poults, and embryonated chicken eggs (ECEs) to examine permissiveness, viral replication kinetics, pathogenesis and pathology. We demonstrated that DF-1 cells in addition to the positive control LLC-PK1 cells are susceptible to SpCoV spike- and RBD- recombinant chimeric virus infections. However, the replication of chimeric viruses in DF-1 cells, but not LLC-PK1 cells, was inefficient. Inoculated 8-day-old turkey poults appeared resistant to icPDCoV-, icPDCoV-SHKU17- and icPDCoV-RBDISU virus infections. In 5-day-old ECEs, significant mortality was observed in PDCoV inoculated eggs with less in the spike chimeras, while in 11-day-old ECEs there was no evidence of viral replication, suggesting that PDCoV is better adapted to cross species infection and differentiated ECE cells are not susceptible to PDCoV infection. Collectively, we demonstrate that the SpCoV chimeric viruses are not more infectious in turkeys, nor ECEs than wild type PDCoV. Therefore, understanding the cell and host factors that contribute to resistance to PDCoV and avian-swine chimeric virus infections may aid in the design of novel antiviral therapies against DCoVs.

Background/Objectives: African swine fever virus (ASFV), the causative agent of African swine fever (ASF), is a highly contagious virus affecting both domestic and feral pig populations with mortality rates approaching 100% within one week of infection. Currently, there are limited treatments or vaccines available to control the disease. Although ASF is endemic in sub-Saharan Africa, the virus has also spread widely, reaching regions of the European Union, Russia, China, Southeast Asia, and, more recently, to the Dominican Republic and Haiti, bringing the threat closer to the United States (U.S.). ASF introduction to the U.S. would have severe consequences for swine producers and the national pork industry. Consequently, there is an urgent need to develop effective vaccine strategies to manage ongoing outbreaks abroad and mitigate the risk of future ASF incursions. Recent efforts have identified several ASFV epitopes and evaluated them in experimental vaccine trials. However, these vaccine candidates have elicited limited protective immune responses and have not demonstrated full protective efficacy. Methods: In this study, we employed in silico modeling and epitope prediction tools to design a synthetic multiepitope ASF protein incorporating key immunogenic regions of ASFV. The goal was to generate a single-antigen construct capable of inducing broad and robust immune responses when formulated with an established nanoparticle-based vaccine platform. The multiepitope ASF protein was subsequently expressed and entrapped into mannose-conjugated chitosan (M-CS) nanoparticles for vaccine formulation. The candidate vaccine, formulated with M-CS nanoparticle-entrapped adjuvant (ADU S100), was administered intramuscularly to pigs, and both T- and B-cell responses were assessed following the primary (DPV 22) and booster (DPV 42) doses. Results: Our M-CS ASF protein vaccine elicited antigen-specific T- and B-cell responses, both of which are recognized as central correlates of protection against ASFV. Conclusions: These promising preliminary immunological findings suggest that this nanoparticle vaccine has the potential to confer protection against ASFV challenge, a hypothesis that will be examined in future studies.

Nonalcoholic fatty liver disease (NAFLD), which shows similar symptoms as fatty liver hemorrhage syndrome (FLHS) in chickens, is the most common cause of chronic liver disease and cancer in humans. NAFLD patients and FLHS in chickens have demonstrated severe liver disorders when infected by emerging strains of human hepatitis E virus (HEV) and avian HEV, respectively. We sought to develop a fatty liver disease chicken model by altering the diet of 3-week-old white leghorn chickens. The high cholesterol, and low choline (HCLC) diet included 7.6% fat with additional 2% cholesterol and 800 mg/kg choline in comparison to 5.3% fat, and 1,300 mg/kg choline in the regular diet. Our diet induced fatty liver avian model successfully recapitulates the clinical features seen during NAFLD in humans and FLHS in chickens, including hyperlipidemia and hepatic steatosis, as indicated by significantly higher serum triglycerides, serum cholesterol, liver triglycerides, cholesterol, and fatty acids. By developing this chicken model, we expect to provide a platform to explore the role of lipids in the liver pathology linked with viral infections and contribute to the development of prophylactic interventions.

Viral hepatitis is primarily caused by five unrelated hepatotropic viruses, hepatitis A, B, C, D, and E. While hepatitis A through C are commonly recognized as causing significant liver disease by general public due to successful public health campaigns, most are not aware of hepatitis E. Despite being unheard of in the public, hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide with an estimated 20 million cases annually. Hepatotropic viruses are notoriously tricky, utilizing differing mechanisms to avoid detection and elimination by the host organism. While hepatitis B and C infections often produce few symptoms in the host while becoming chronic and spreading silently to new hosts, HEV utilizes a different strategy to continue circulating in its hosts. HEV’s long incubation period and ability to self-resolve in many infected individuals coupled with animal reservoirs that show little disease upon infection allow HEV to transmit to humans through the food chain. Endemic human strains have similar strategies, circulating at low levels within the populace waiting for conditions associated with socio economic turmoil when sanitary conditions decrease allowing for massive outbreaks through contaminated water. This virological game of hide and seek ensures the continued survivability and transmission of the pathogen. While most otherwise healthy individuals will be able to self-resolve HEV infections, people with underlying comorbidities, immunocompromised individuals, and pregnant women in their third trimester are at much greater risk of succumbing to hepatitis E. There is still much work to be done to unravel the nuances of HEV’s deadly hide-and-seek game so that humans may rid themselves of this malady. Recently, the new emerging zoonotic rat HEV has been a hot topic in the field of HEV. The first rodent-associated hepevirus was discovered in 2010 from fecal and liver specimens of rats in Germany. Based on sequence divergence, this new virus was classified as a separate species from the primary human pathogen Paslahepevirus balayani (Previously Orthohepevirus A) and named Orthohepevirus C which recently changed to Rocahepevirus ratti, (common name - rat HEV) genotype HEV-C1 and C2. Since then, Rocahepevirus ratti HEV-C1 has been detected in multiple wild rat species, including in wild Norway rats (Rattus norvegicus) in Germany. Rocahepevirus ratti (HEV-C1) has been detected in rats in 12 European countries (Austria, Belgium, Czech Republic, Denmark, France, Germany, Greece, Hungary, Italy, Lithuania, Spain, and Switzerland). It has been found in Norway and in Black rats (Rattus rattus) in the USA and Indonesia, Tanezumi rats (Rattus tanezumi) in Vietnam, Bandicoot rats (Bandicota indica), Buff-breasted rats (Rattus flavipectus), and the Lesser ricefield rat (Rattus losea) in China. These studies suggest that HEV-C1 is geographically widespread among rats and present on a minimum of three continents (Asia, America, and Europe). Interestingly, according to a Chinese study, Asian musk shrews (Suncus murinus) also seem to be a reservoir of HEV-C1 because of its cohabitation with rats. Rat HEV-C1 was also identified from a Syrian brown bear (Ursus arctos syriacus) in a zoo animal in Germany, most likely the result of a spillover infection from rats, based on the close phylogenetic relationship with the wild rat HEV-C1 also described in Germany. Rat HEV is also present in other small mammals and rodents besides rats. HEV-C2 was discovered in ferrets and mink and recent reports describe further unassigned, genetically novel rodent rat HEV strains from Chevrier’s field mouse (Apodemus chevrieri), Pere David’s vole (Eothenomys melanogaster), related to the first identified vole-borne rat hepevirus strain in this group and from carnivorous bird species, kestrel (Falco tinnunculus), and falcon (Falco peregrinus) possibly due to its diet. The first confirmed human infection associated with rat hepevirus was detected in Hong Kong, in 2017. The patient was a 56-year-old man who had undergone a liver transplant and was immunocompromised. He had abnormal liver function tests at day 59 post-transplantation, and only RT-PCR using pan-orthohepevirus primers and sequencing could confirm the presence of rat origin HEV-C1 in plasma, feces, saliva, and liver tissue specimens. The patient was successfully treated with a 7-month-long oral ribavirin therapy. The source of the infection was potentially associated with rodent droppings in his living environment. While the transmission of rat HEV to humans is not yet fully understood, it is believed that humans may have been infected directly via contact with environmental surfaces contaminated by rat droppings or indirectly by contamination of water or food products (e.g., consumption of rat meat or adulterated meat products). The transmission of rat HEV from rats (or rodents) through an intermediate animal host (e.g., agricultural animals, etc.) to humans is an open question. Mapping the transmission routes of rat HEV is necessary to prevent future human infections. In general, zoonotic HEV has been shown to be linked with a higher rate of pregnancy mortality in humans. Whether rat HEV, which has been recently found to be zoonotic, has similar pregnancy pathogenesis capability needed to be studied.The major reservoir of Paslahepevirus balayani is swine that are known to transmit Paslahepevirus gt3 and gt4 to humans. In response to the increase in human infection by rat HEV (21 cases), I sought to construct and characterize an infectious cDNA clone of rat HEV to understand whether swine may serve as a transmission host for rat HEV. The complete genome of rat HEV strain LCK-3110 was cloned downstream of a bacteriophage SP6 promoter. Capped genomic RNA was generated by in vitro transcription. Direct intrahepatic inoculation of gnotobiotic pigs with capped RNA transcripts was performed to study the replication competence of transcripts of rat HEV. Ten percent fecal suspension of intestinal contents derived from the HEV positive gnotobiotic pigs was intravenously inoculated via ear vein in conventional pigs. Sentinel pigs were comingled with the rat HEV inoculated conventional pigs at 7 days post inoculation (DPI). Separate pig groups were inoculated with Paslahepevirus balayani human HEV (US-2) strain and phosphate buffered saline (PBS), as positive and negative controls, respectively. Transcripts of rat HEV developed active HEV infection as evidenced by viremia and fecal virus shedding in gnotobiotic pigs. The infectivity was further confirmed by the successful infection of conventional pigs via intravenous injection of purified 10% fecal suspension and subsequent infection of naive sentinel pigs as shown by seroconversion, viremia, fecal virus shedding, and immunohistochemistry. Our results indicate that the LCK-3110 strain of rat HEV is capable of cross-species infection in pigs. Rat HEV has an expanding host range, including pigs, that could be a transmission source to humans.Our studies continued by experimentally testing another agricultural important species, chicken, for their permissiveness to rat-HEV. I initiated the in vitro experiments by performing transfection of the chicken liver cell lines with in vitro transcribed capped viral RNA and replication was assessed. The LCK-3110 strain of rat HEV was capable of replicating in chicken liver cell lines. Moreover, cross species transmission ability in chickens was studied via intrahepatic inoculation of specific pathogen free chickens with capped RNA transcripts of the LCK-3110 rat-HEV strain. RT-qPCR for rat HEV was performed weekly in serum, fecal, liver, and bile samples. Mild infection was seen in chicken as demonstrated by fecal viral RNA shedding, viremia, histopathology, and viral protein detected by immunohistochemistry of liver samples. Thus, rat HEV is an emerging infectious virus with a broad host range and can spillover across multiple species including chickens.Paslahepevirus balayani strains are known for their detrimental effects during pregnancy by causing pregnancy related disorders. Recent findings have demonstrated the ability of the Paslahepevirus balayani to replicate within placental cell lines suggesting a direct effect on the placenta and fetus. To study whether zoonotic rat HEV strains possess a similar host placental tropism, we utilized JEG-3 cells to understand the replicative ability of an infectious clone of a recently reported rat HEV, the LCK-3110 strain. Infectious cDNA clones of Pasla-, Avi-, and Roca- hepeviruses were transcribed, and then transduced into JEG-3 cells. Pasla- and Avi-hepeviruses were used as controls in the study. Cells were harvested, and cell lysates were used for testing infectivity. Five days post transfection or after inoculation onto naive HepG2/C3A cells, the cells were analyzed for infection. Replication in transduced JEG-3 cells and infection potential in HepG2/C3A cells was assessed via an indirect immunofluorescence assay and a flow cytometry assay. We found that the genetically diverse Rocahepevirus ratti LCK-3110 strain did not have efficient replication in JEG-3 cell cultures. Our results suggest that current zoonotic rat HEV strains may not be able to cause pregnancy mortality in humans.Overall, my research has added to the HEV knowledge base with the novel findings, a new infectious clone, and potentially opening a new area of research. Rat HEV infection in both immunocompetent and immunosuppressed individuals with an undefined transmission source made the study an urgent need to assess a possible transmission route to humans. My thesis enhances knowledge on the potential spread of rat HEV through agriculturally important species: pigs and chickens. Experimental inoculation as well as natural infection in sentinel pigs and chickens highlights the ability of rat HEV to transmit and spread through a natural fecal-oral route. My study demon

Abstract Strains of Rocahepevirus ratti, an emerging hepatitis E virus (HEV), have recently been found to be infectious to humans. Rats are a primary reservoir of the virus; thus, it is referred to as “rat HEV”. Rats are often found on swine farms in close contact with pigs. Our goal was to determine whether swine may serve as a transmission host for zoonotic rat HEV by characterizing an infectious cDNA clone of a zoonotic rat HEV, strain LCK-3110, in vitro and in vivo. RNA transcripts of LCK-3110 were constructed and assessed for their replicative capacity in cell culture and in gnotobiotic pigs. Fecal suspension from rat HEV-positive gnotobiotic pigs was inoculated into conventional pigs co-housed with naïve pigs. Our results demonstrated that capped RNA transcripts of LCK-3110 rat HEV replicated in vitro and successfully infected conventional pigs that transmit the virus to co-housed animals. The infectious clone of rat HEV may afford an opportunity to study the genetic mechanisms of rat HEV cross-species infection and tissue tropism.